1. Field of the Invention
The present invention is directed to an improvement in the assay art. It relates to methods and compositions for inhibiting endogenous enzyme (e.g., catalase) activity in biological fluids, and more specifically to inhibiting hydrogen peroxide-degrading enzyme activity in biological fluids during qualitative or quantitative analysis of biological fluids, when hydrogen peroxide participates in a signal-generation reaction.
2. Description of the Background Art
Human serum contains many enzymes such as catalase, glutathione reductase and peroxidase, all of which readily decompose hydrogen peroxide to oxygen and water. The most important of these enzymes is serum catalase [EC 1.11.1.6]. The amount of catalase that is present in the plasma of normal human subjects can degrade hydrogen peroxide at a rate of 0.01 to 0.05 moles/liter/minute (Yamagata et al., Tohoku J. Exp. Med. 57: 101-107, 1953; Goth et al., Clin. Chem. 29: 741-743, 1983). Catalase is also present in erythrocytes in levels 3600 fold higher than in plasma, giving erythrocytes the capacity to degrade gram quantities of hydrogen peroxide over several minutes, Gaetani et al., Blood 73: 334-339 (1989).
In fact, catalase has been used to verify the specificity of assays of hydrogen peroxide in plasma or blood, and the degradation of hydrogen peroxide or inhibition of the assay system by the sample should be checked, as catalase present in blood or plasma can reduce the value of measured hydrogen peroxide to zero, Nahum et al., Free Radical Biology & Medicine 6: 470-484, 1989.
The ability to measure a wide variety of physiologically active compounds, both naturally occurring and synthetic, has become of increasing importance, both as an adjunct to diagnosis and as therapy. While for the most part assays of physiological fluids and drugs have required clinical laboratory determinations, there is an increasing awareness of the importance of being able to conduct assays in a physician's office and/or in the home. To be able to perform an assay outside of a clinical laboratory setting requires that an assay have a simple protocol and be relatively free of sensitivity to small changes in the condition under which the assay is conducted. Although a number of systems have been developed to try to address the problems associated with analysis outside of a clinical laboratory, there is nevertheless a continuing interest in providing improved and alternative methods to those which are presently generally available.
If a clinical detection reaction requires the presence of hydrogen peroxide at any stage of the assay, any endogenous serum catalase activity present in the reaction system will interfere with the reaction. Dilution of the sample to reduce catalase interference necessarily reduces the sensitivity of the reaction. Many clinical detection systems require the presence of hydrogen peroxide, including determination of cholesterol, triglycerides, glucose, ethanol, lactic acid, etc. For example, when free cholesterol is oxidized by cholesterol oxidase to form cholesteneone and hydrogen peroxide, the hydrogen peroxide so generated can be used to measure the amount of cholesterol originally in the sample.
One of the primary needs in clinical assays is the need to determine cholesterol or triglyceride, including high and low density lipoprotein, levels in blood. There is a clear relationship between total blood cholesterol (mainly the LDL fraction) and coronary artery disease (Journal of the American Medical Association 253: 2080-2086, 1985). New guidelines have been established for adults over 20 years of age to identify risk groups associated with blood cholesterol levels, wherein less than 200 mg/dl is a desirable blood cholesterol; 239 mg/dl is borderline high blood cholesterol, and more than 240 mg/dl is considered to be high blood cholesterol and thus the patient is considered to be at high risk.
Because cholesterol levels can be controlled by both diet and cholesterol lowering drugs for those patients at risk, the ability to monitor one's own cholesterol at home for those individuals at risk provides an important tool for monitoring cholesterol levels and thus reducing the potential for heart disease. The ability to measure other naturally occurring compounds of physiological importance, as well as levels of synthetic drugs or hormones, is also of great interest.
Detection of components in liquids, such as cholesterol in blood, by test strips is well known. Vogel et al., in U.S. Pat. No. 4,312,834, and Goodhue et al., in U.S. Pat. No. 3,983,005, disclose test strips which can be used for detecting cholesterol in serum. Problems associated with catalase activity in human serum are avoided by including competing enzymes such as peroxidases in large excess so that the effects of catalase in the sample are minimized.
Allen et al., in U.S. Pat. No. 4,999,287, disclose stripsticks for direct assay of physiologically active compounds such as cholesterol wherein the adverse effects of catalase are lessened by diluting the sample.
In reaction systems where undiluted plasma samples are incorporated, the activity of endogenous enzymes may interfere with an analyte or precursor necessary to measure an analyte of interest. Additionally, many enzymes which are used as reagent in clinical assays may be contaminated with catalase or with other enzymes that destabilize hydrogen peroxide. In chemical reactions that utilize hydrogen peroxide to oxidize a dye or other intermediate to generate a colored or other type of indicator species, the stability of hydrogen peroxide is an absolute necessity in providing an accurate measurement of the analyte of interest.
Enzymes have been incorporated into detergent compositions because of the enzymes' effectiveness against a variety of common stains which are fixed to textiles and laundry. In particular, proteolytic enzymes, which possess the ability to digest and degrade proteinaceous matter, are used to remove from textile proteinic strains such as blood, perspiration milk, cocoa, gravy and other types of sauces. However, many of these detergent compositions also include peroxide and/or persulfate bleaching compounds, and catalase, which is present in many of these common stains, including blood, readily destroys these peroxide and persulfate bleaching compounds.
In order to minimize the effect of catalase on the bleaching compounds, inhibitor compounds can be incorporated in the detergent compositions, as disclosed in Gobert, U.S. Pat. No. 3,751,222; Gobert et al., U.S. Pat. No. 3,606,990, and Oukadi et al., U.S. Pat. No. 4,753,750. The inhibitors may be contacted with the stained cloth during a soaking or prewashing stage prior to contact with the peroxide or persulfate bleaching agent or, alternately, during the washing step, the inhibitors may be included with the peroxide or persulfate bleaching agent. Of particular importance are compositions which comprise a mixture of inhibitor and detergent. These inhibitors are designed to be used in the presence of surfactants, including anionic, nonionic, amphoteric and cationic surfactants in the presence of peroxide and/or persulfate bleaching agents. These bleaching compositions perform best at high pH values, permitting their use in conjunction with common household laundry soaps and detergents.
Among the inhibitors which have been found to be useful in detergent compositions for inhibiting the activity of catalase in soaking or washing solutions are hydroxylamine sulfate; hydroxylamine hydrochloride; phenylhydrazine; hydrazine; hydrazine sulfate; saturated phenols; polyphenols substituted with at least one of NH.sub.2, SO.sub.2 NH.sub.2, Cl, Br, NO.sub.2 ; aminophenols including o-amino-p-chlorophenol; aminotriazoles; alkali metal chlorate; sodium nitride; alkali metal cyanurates; and mixtures of the above.
Many of the above inhibitors used in detergent compositions are active only at extreme pH values, such as at less than 4.0 or greater than 12.0, depending upon the type of detergent and/or bleach composition used. The useful pH range in Gobert et al. is generally from 8 to 11, and preferably from 9 to 10.5. Additionally, many of these compounds are strong reducing agents, such as formaldehyde, which are not compatible with biological assay systems. Others of the inhibitors which are strong oxidizing agents, including alkali metal hypochlorites, alkali metal salts of chlorocyanuric acids, or potassium salts of monopersulfuric acid, may be strong denaturing agents. There properties may be acceptable in detergent formulations, but they are completely unacceptable in clinical assay systems in which the components of the systems are to be preserved, not destroyed.
Additionally, heavy metal salts such as mercuric chloride, nickel salts, or solvents such as acetone, either precipitate proteins or interfere with enzyme activity site activity. Thus, many of these inhibitors cannot be used in diagnostic enzymatic reactions, in which the substances in the samples are not stable at extremes of pH or in the presence of many types of inhibitor compounds.
Many of the conventional tests which require the presence of hydrogen peroxide at some stage of the assay depend upon the use of peroxidase for detection of hydrogen peroxide. However, specific inhibitors for peroxidase include cyanides, sulfides, fluorides, and azides. Therefore, in order to reduce the effects of endogenous catalase in samples from decomposing the hydrogen peroxide to be detected in an assay, it is important not to use catalase inhibitors based upon cyanides, sulfides, fluorides, or azides.
Other workers have inhibited the action of catalase on hydrogen peroxide for different reasons. For example, Fujie et al. in U.S. Pat. No. 5,055,398, disclose a clinical assay for contents of bodily fluid wherein a sample of bodily fluid is first processed with catalase for decomposing hydrogen peroxide that may be present in a fluid sample. That is, catalase is first added to the sample from outside of the system specifically to decompose any hydrogen peroxide that may be present in the sample. Then, because catalase in the sample may interfere with subsequent assays, Fujie et al. add an inhibitor for catalase, such as sodium azide, hydrogen cyanide, hydrogen sulfide, ammonium hydroxide, or 3- amino-1,2,4-triazole, to inhibit the catalase. Unfortunately, many of these inhibitors, including hydrogen cyanide, hydrogen sulfide, and sodium azide are compounds which have been found to decompose horseradish peroxidase, so that the Fujie et al. method is limited in use to those assays which do not subsequently use peroxidase.
It is also known that different peroxidase isoenzymes are inhibited to different degrees by inhibitors, Kay et al., J. of Biol. Chem., 242 (10): 2470-2473, 1967. Thus, the inhibitory effect of an enzyme inhibitor may vary depending upon the particular isoenzyme used, and may also vary depending upon the reaction milieu.
Beaucamp et al., in U.S. Pat. No. 3,925,164, disclose a method for determination of cholesterol by using catalase as a reagent to detect hydrogen peroxide formed by treating cholesterol with cholesterol oxidase. In this case, catalase is added as a reagent for detecting hydrogen peroxide formed; there is certainly no reason that catalase would be considered to interfere with the assay reactions in this case, as the catalase is used as a reagent.
Kano et al., in U.S. Pat. No. 3,862,885, teach a process for determining uric acid in a blood sample with uricase using a catalase inhibitor such as sodium azide. As discussed above, however, use of sodium azide to inhibit the action of catalase precludes later use of peroxidases in the assay. Since many clinical assays depend upon the production or use of hydrogen peroxide with peroxidase, the use of sodium azide to inhibit catalase introduces other errors into the assay.
No admission is made that any of the background references cited above constitute prior art or pertinent prior art.